The long term objective of this proposal is to study the cell biology of hormone secretion by endocrine cells. AtT-20 cells, which secrete adrenocorticotropic hormone (ACTH) are a very well characterized cellular model for regulated secretion. Rab proteins are a class of small GTP-ases that seem to be key regulators of vesicular traffic. A family of Rabs, the Rab3 isoforms, associate with the regulated exocytotic pathway. Each Rab3 isoform may be associated at different stages of the ACTH secretory pathway. Understanding the distribution of different Rab3 proteins in AtT-20 cells will help in the identification of different steps in the biogenesis and maturation of dense core granules. AtT-20 cells express both Rab3A and Rab3D which are localized in different cellular compartments. The first aim of this proposal is to determine the localization of Rab3D and Rab3A in the ACTH pathway by cell fractionation and electron microscopy techniques. In AtT-20 cells expressing mutated Rab3D N135I, ACTH-containing granules are able to position near the plasma membrane and to undergo regulated exocytosis. Dense core granules formed in cells expressing mutated Rab3D may be blocked at an early stage of maturation and lack fundamental components necessary for their release. The second aim of this proposal is to identify these components. Rab proteins shuttle in between a GTP-bound, activated conformation and a GDP-bound, inactive form. Rab3D N135I is presumably locked in one rigid conformation and may sequester a protein (Rab3D-binding protein) which functions as a downstream effector of Rab3D. Another possibility is that mutated Rab3D depletes factors which modulate Rab3 protein activity of Rab3 binding to membranes. The third aim of this proposal is to identify and characterize these factors. Endocrine diseases such as Diabetes Mellitus type I and Cushing Disease are due, respectively, to a lack of inulin and increased concentrations of ACTH hormone into the blood. The long- term goal of this proposal is to help understanding how cells secrete hormones in a regulated fashion and to set novel therapeutic approaches for patients with endocrine diseases.